Participation of kin17 protein in replication factories and in other DNA transactions mediated by high molecular weight nuclear complexes

The Homo sapiens kin17 ((HSA)kin17) protein is a chromatin-associated protein conserved during evolution and overproduced in certain human tumor cell lines. For the first time, immunoelectron microscopy analysis of endogenous (HSA)kin17 protein revealed an ultrastructural co-localization of (HSA)kin17 and bromodeoxyuridine (BrdUrd) at sites of DNA replication after either short (15 min) or long (120 min) pulses of BrdUrd labeling. After hydroxyurea (HU) or L-mimosine (Mimo) block and withdrawal, we observed that (HSA)kin17 was recruited onto the chromatin during the re-entry and the progression in the S phase. These results are consistent with a major role of (HSA)kin17 protein in DNA replication factories. Other treatments hampering replication fork progression and/or inducing double-strand breaks also triggered an accumulation and a concentration of the chromatin-bound (HSA)kin17 protein into large intranuclear foci 24 h post-treatment. Moreover, HU- and Mimo-induced (HSA)kin17 foci were retained in the nucleus after detergent extraction, suggesting a strong association with nuclear structures. Gel filtration analyses of cellular extracts showed that endogenous (HSA)kin17 protein co-eluted with both replication proteins RPA32 and RPA70 in a fraction containing complexes of M(r) 600,000. Interestingly, HU-induced G(1)-S arrest triggered an increase in the molecular weight of complexes containing (HSA)kin17 protein. Hence, treatments interfering with either initiation and/or elongation of DNA replication also recruited chromatin-bound (HSA)kin17 protein. We hypothesize that in the presence of unrepaired DNA damage, (HSA)kin17 protein concentrated into high molecular weight complexes probably to create a bridge that contributes to the harmonization of DNA replication and repair.     

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.     

Development of new EBV-based vectors for stable expression of small interfering RNA to mimick human syndromes: application to NER gene silencing

We developed and characterized replicative small interfering RNA (siRNA) vectors for efficient, specific, and long-term gene silencing in human cells. We created stable XPA(KD) and XPC(KD) (knockdown) syngeneic cell lines to mimic human cancer-prone syndromes. We also silenced (HSA)KIN17. Several clones displaying undetectable protein levels of XPA, XPC, or (HSA)kin17 were grown for more than 300 days. This stability of gene silencing over several months of culture allows us to assess the specific involvement of these proteins in UVC sensitivity in syngeneic cells. Unlike XPA, (HSA)KIN17, and XPC gene silencing dramatically impeded HeLa cell growth for several weeks after transfection. As expected, XPA(KD) and XPC(KD) HeLa cells were highly UVC sensitive. They presented an impaired unscheduled DNA synthesis after UVC irradiation. Interestingly, XPC(KD) HeLa clones were more sensitive to UVC than their XPA(KD) or KIN17(KD) counterparts. Hygromycin B withdrawal led to the total disappearance of EBV vectors and the resumption of normal XPA or XPC protein levels. Whereas reverted XPA(KD) cells recovered a normal UVC sensitivity, XPC(KD) cells remained highly sensitive, suggestive of irreversible damage following long-term XPC silencing. Our results show that in HeLa cells, (HSA)kin17 participates indirectly in early events following UVC irradiation, and XPC deficiency strongly affects cell physiology and contributes to UVC sensitivity to a greater extent than does XPA. EBV-based siRNA vectors improve the interest of siRNA by permitting long-term gene silencing without the safety concerns inherent in viral-based siRNA vehicles.     

Up-regulation of kin17 is essential for proliferation of breast cancer

Background

Kin17 is ubiquitously expressed at low levels in human tissue and participates in DNA replication, DNA repair and cell cycle control. Breast cancer cells are characterized by enabling replicative immortality and accumulated DNA damage. However, whether kin17 contributes to breast carcinogenesis remains unknown.

Methodology/Principal Findings

In this study, we show for the first time that kin17 is an important molecule related to breast cancer. Our results show that kin17 expression was markedly increased in clinical breast tumors and was associated with tumor grade, Ki-67 expression, p53 mutation status and progesterone receptor expression, which were assessed in a clinicopathologic characteristics review. Knockdown of kin17 inhibited DNA replication and repair, blocked cell cycle progression and inhibited anchorage-independent growth, while increasing sensitivity to chemotherapy in breast cancer cells. Moreover, kin17 silencing decreased EGF-stimulated cell growth. Furthermore, overexpression of kin17 promoted DNA replication and cell proliferation in MCF-10A.

Conclusions/Significance

Our findings indicate that up-regulation of kin17 is strongly associated with cellular proliferation, DNA replication, DNA damage response and breast cancer development. The increased level of kin17 was not only a consequence of immortalization but also associated with tumorigenesis. Therefore, kin17 could be a novel therapeutic target for inhibiting cell growth in breast cancer.     

Cell cycle progression in the presence of irreparable DNA damage is controlled by a Mec1- and Rad53-dependent checkpoint in budding yeast.

We studied the response of nucleotide excision repair (NER)-defective rad14Delta cells to UV irradiation in G(1) followed by release into the cell cycle. Only a subset of checkpoint proteins appears to mediate cell cycle arrest and regulate the timely activation of replication origins in the presence of unrepaired UV-induced lesions. In fact, Mec1 and Rad53, but not Rad9 and the Rad24 group of checkpoint proteins, are required to delay cell cycle progression in rad14Delta cells after UV damage in G(1). Consistently, Mec1-dependent Rad53 phosphorylation after UV irradiation takes place in rad14Delta cells also in the absence of Rad9, Rad17, Rad24, Mec3 and Ddc1, and correlates with entry into S phase. Two-dimensional gel analysis indicates that late replication origins are not fired in rad14Delta cells UV-irradiated in G(1) and released into the cell cycle, which instead initiate DNA replication from early origins and accumulate replication and recombination intermediates. Progression through S phase of UV-treated NER-deficient mec1 and rad53 mutants correlates with late origin firing, suggesting that unregulated DNA replication in the presence of irreparable UV-induced lesions might result from a failure to prevent initiation at late origins.     

Selective interactions of human kin17 and RPA proteins with chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner

Several proteins involved in DNA synthesis are part of the so-called 'replication factories' that are anchored on non-chromatin nuclear structures. We report here that human kin17, a nuclear stress-activated protein, associates with both chromatin and non-chromatin nuclear structures in a cell cycle- and DNA damage-dependent manner. After L-mimosine block and withdrawal we observed that kin17 protein was recruited in the nucleus during re-entry and progression through S phase. These results are consistent with a role of kin17 protein in DNA replication. About 50% of the total amount of kin17 protein was detected on nuclear structures and could not be released by detergents. Furthermore, the amount of kin17 protein greatly increased in both G(1)/S and S phase-arrested cells in fractions containing proteins anchored to nuclear structures. The detection of kin17 protein showed for the first time its preferential assembly within non-chromatin nuclear structures in G(1)/S and S phase-arrested cells, while the association with these structures was found to be less stable in the G(2)/M phase, as judged by fractionation of human cells and immunostaining. In asynchronous growing cells, kin17 protein interacted with both chromatin DNA and non-chromatin nuclear structures, while in S phase-arrested cells it interacted mostly with non-chromatin nuclear structures, as judged by DNase I treatment and in vivo UV cross-linking. In the presence of DNA damage in S phase cells, the distribution of kin17 protein became mainly associated with chromosomal DNA, as judged by limited formaldehyde cross-linking of living cells. The physical interaction of kin17 protein with components of the nuclear matrix was confirmed and visualized by indirect immunofluorescence and immunoelectron microscopy. Our results indicate that, during S phase, a fraction of the human kin17 protein preferentially associates with the nuclear matrix, a fundamentally non-chromatin higher order nuclear structure, and to chromatin DNA in the presence of DNA damage.     

The catalytic subunit DNA-dependent protein kinase (DNA-PKcs) facilitates recovery from radiation-induced inhibition of DNA replication

Exposure of cells to ionizing radiation inhibits DNA replication in a dose-dependent manner. The dose response is biphasic and the initial steep component reflects inhibition of replicon initiation thought to be mediated by activation of the S-phase checkpoint. In mammalian cells, inhibition of replicon initiation requires the ataxia telagiectasia mutated (ATM) gene, a member of the phosphatidyl inositol kinase-like (PIKL) family of protein kinases. We studied the effect on replicon initiation of another member of the PI-3 family of protein kinases, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) by measuring either total DNA synthesis, or size distribution of nascent DNA using alkaline sucrose gradient centrifugation. Exposure of human cells proficient in DNA-PKcs (HeLa or M059-K) to 10 Gy inhibited replicon initiation in a time-dependent manner. Inhibition was at a maximum 1 h after irradiation and recovered at later times. Similar treatment of human cells deficient in DNA-PKcs (M059-J) inhibited replicon initiation to a similar level and with similar kinetics; however, no evidence for recovery, or only limited recovery, was observed for up to 8 h after irradiation. In addition a defect was observed in the maturation of nascent DNA. Similarly, a Chinese hamster cell line deficient in DNA-PKcs (irs-20) showed little evidence for recovery of DNA replication inhibition up to 6 h after irradiation, whereas the parental CHO cells showed significant recovery and an irs-20 derivative expressing the human DNA-PKcs complete recovery within 4 h. Normal kinetics of recovery were observed in xrs-5 cells, deficient in Ku80; in 180BR cells, deficient in DNA ligase IV; as well as XR-1 cells, deficient in XRCC4, an accessory factor of DNA ligase IV. Since all these cell lines share the DNA double strand break rejoining defect of M059-J and irs20 cells, the lack of recovery of DNA replication in the latter cells may not be attributed entirely to the prolonged presence of unrepaired DNA dsb. We propose that DNA-PKcs, in addition to its functions in the rejoining of DNA dsb and in DNA replication, also operates in a pathway that in normal cells facilitates recovery of DNA replication after irradiation.     

The relationship between the cell time available for repair and the effectiveness of a damaging treatment in provoking the formation of sister-chromatid exchanges

The effectiveness of a given dosage of visible light in inducing increased yields of SCEs was studied in Allium cepa L. meristems. Cells were first grown for one cycle time in the presence of BrdUrd and then irradiated at different times throughout the second cell cycle. The effectiveness of this treatment in provoking the formation of SCEs increases the closer the irradiation time is to the beginning of the S phase, and then decreases rapidly as cells progress through the S period. The largest increase in SCEs is obtained when irradiation coincides with early S phase. These results strongly suggest that SCEs arise at the time of DNA replication due to the presence of unrepaired lesions. Since repair appears to be a time-dependent process, the shorter the interval between damage induction and DNA replication, the greater the number of lesions that remain unrepaired, and as a consequence, the higher the effectiveness of the damaging treatment in provoking the formation of SCEs.     

染色质改构因子BRG1降低UV照射引起的细胞凋亡

生命体的遗传物质基础是DNA分子,多种因素可以作用于细胞内的DNA分子,导致多种类型的DNA损伤。若受损的DNA得不到及时和有效的修复,细胞将走向凋亡或发生变异。染色质改构复合物(chromatin remodeling complex)在基因表达调控和DNA复制等方面扮演着重要角色。依赖ATP的染色质改构复合物SWI/SNF的核心亚基Brahma Related Gene1(BRG1)在染色质结构调整和基因转录调控等多个细胞进程中具有重要作用,仅有有限的文献报道BRG1参与到DNA的损伤修复过程。因此,进一步研究与验证BRG1在调控DNA的损伤修复进而挽救细胞凋亡中的作用十分重要。本文通过利用不同强度的UV照射检测细胞凋亡的情况,初步建立了DNA损伤修复的实验体系。将BRG1表达质粒瞬时转染到SW13(BRG1-/-)细胞系中,并利用30J/m2的UV照射,分别在0h、6h和24h检测细胞早期凋亡程度。结果表明,SW13(BRG1-/-)细胞中瞬时表达BRG1可以明显降低由UV照射引起的细胞凋亡,其中UV照射后24h的细胞表现最明显。我们进一步在HeLa细胞中通过瞬时表达BRG1验证了上述结果。由于BRG1通过染色质改构在基因的转录调控、复制和重组等方面起着重要的作用,我们推测BRG1可能通过染色质改构参与了DNA的损伤修复过程,进而影响了细胞凋亡。     

Trans replication and high copy numbers of wheat dwarf virus vectors in maize cells.

The replication of shuttle vectors derived from Wheat Dwarf Virus, a monopartite geminivirus, was studied in cultured maize endosperm cells, and in the Black Mexican Sweet (BMS) maize cell line. Using in vivo labeling and DNA methylation analysis, we showed that replication was initiated within 24 hrs after transfection, and did not require cell division in both cell lines. Copy numbers of 30,000 ds DNA molecules per cell were observed in endosperm cells after three days. The replication protein was shown to act in trans, since the wild type gene of the shuttle vector enabled replication-deficient vectors carrying mutated genes to replicate. These properties suggest that WDV may have similar applications in plants as SV40 in mammalian cells.     

DNA repair in human xeroderma pigmentosum group C cells involves a different distribution of damaged sites in confluent and growing cells.

Xeroderma pigmentosum is a human disease consisting of several complementation groups that are deficient in excision repair. Group C is one in which excision repair occurs at about 20-30% of normal levels. The distribution of mended sites in relation to unrepaired sites has been determined by cutting remaining unrepaired pyrimidine dimers with Microccocus luteus UV endonuclease. The mended sites have been found clustered together in a fashion that depended on cell proliferation. In confluent group C cells, the mended sites were clustered in regions where dimer excision was as efficient as excision in the DNA of normal cells. In proliferating group C cells, however, mended sites were randomly dispersed. The total amount of repair replication was the same in confluent and proliferating cells. Since previous work has shown that confluent group C cells show more extensive recovery from the lethal effects of UV irradiation than some other groups, clustered repair may correlate with a more efficient mechanism of restoring cell viability. The different distribution of repaired sites during DNA replication may be the result of changes in the state of the substrate for repair or changes in the metabolic priorities of DNA polymerases.     

Sequestration of Mammalian Rad51-Recombination Protein into Micronuclei

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after γ irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.     

Involvement of DNA-dependent protein kinase in UV-induced replication arrest.

Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G(1)/S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV-induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs(+))), as measured by [(3)H]thymidine incorporation, was significantly arrested by 4 h following UV irradiation, whereas M059J (DNA-PKcs(-)) cells were much less affected. Similar results were obtained with the in vitro replication reactions where immediate replication arrest occurred in DNA-PKcs(+) cells following UV irradiation, and only a gradual decrease in replication activity was observed in DNA-PKcs(-) cells. Reversal of replication arrest was observed at 8 h following UV irradiation in DNA-PKcs(+) cells but not in DNA-PKcs(-) cells. Reversal of UV-induced replication arrest was also observed in vitro by the addition of a DNA-PK inhibitor, wortmannin, or by immunodepletion of DNA-PKcs, supporting a positive role for DNA-PK in damage-induced replication arrest. The RPA-containing fraction from UV-irradiated DNA-PKcs(+) cells poorly supported DNA replication, whereas the replication activity of the RPA-containing fraction from DNA-PKcs(-) cells was not affected by UV, suggesting that DNA-PKcs may be involved in UV-induced replication arrest through modulation of RPA activity. Together, our results strongly suggest a role for DNA-PK in S-phase (replication) arrest in response to UV irradiation.     

Effect of somatostatin on repair of ionizing radiation-induced DNA damage in pituitary adenoma cells GH3

Ionizing radiation and somatostatin analogues are used for acromegaly treatment to achieve normalization or reduction of growth hormone hypersecretion and tumor shrinkage. In this study, we investigated a combination of somatostatin (SS14) with ionizing radiation of (60)Co and its effect on reparation of radiation-induced damage and cell death of somatomammotroph pituitary cells GH3. Doses of gamma-radiation 20-50 Gy were shown to inhibit proliferation and induce apoptosis in GH3 cells regardless of somatostatin presence. It has been found that the D(0) value for GH3 cells was 2.5 Gy. Somatostatin treatment increased radiosensitivity of GH3 cells, so that D(0) value decreased to 2.2 Gy. We detected quick phosphorylation of histone H2A.X upon irradiation by the dose 20 Gy and its colocalization with phosphorylated protein Nbs-1 in the site of double strand break of DNA (DSB). Number of DSB decreased significantly 24 h after irradiation, however, clearly distinguished foci persisted, indicating non repaired DSB, after irradiation alone or after combined treatment by irradiation and SS14. We found that SS14 alone triggers phosphorylation of Nbs1 (p-Nbs1), which correlates with antiproliferative effect of SS14. Irradiation also increased the presence of p-Nbs1. Most intensive phosphorylation of Nbs1 was detected after combined treatment of irradiation and SS14. The decrease of the number of the DSB foci 24 h after treatment shows a significant capacity of repair systems of GH3 cells. In spite of this, large number of unrepaired DSB persists for 24 h after the treatment. We conclude that SS14 does not have a radioprotective effect on somatomammotroph GH3 cells.     

Early Syncytium Formation by Bovine Leukemia Virus

Bovine leukemia virus (BLV) from either persistently infected bat cells or fetal lamb kidney cells induced rapid syncytium formation in F81 indicator cells. Distinct syncytia were seen within 2 h after inoculation of cells with highly concentrated (500-fold) cell-free BLV preparations and within 4 to 8 h when unconcentrated cell-free BLV preparations were used. Indicator cell densities of 1 x 10(5) to 2 x 10(5) were optimal for rapid and maximal syncytium formation. Pretreatment of BLV with reference BLV leukemic serum and antiserum prepared against purified BLV significantly inhibited (95%) syncytium formation. Reference bovine viral diarrhea virus serum, foamy-like bovine syncytial virus serum, and control serum had little effect (17% inhibition). Antiserum to BLV gp51 inhibited syncytium formation by greater than 96%, whereas antiserum to BLV p24 reduced syncytium activity to a much lesser extent (38% inhibition). Treatment of BLV with beta-propiolactone (0.005 to 0.05%) had little or no effect upon syncytium-forming activity, whereas UV irradiation (15 ergs/mm(2) per s for 30 min) reduced, but did not completely destroy, the fusion activity. However, both beta-propiolactone and UV irradiation drastically reduced the replication potential of BLV, as demonstrated by the lack of p24 expression in the inoculated cells. Concentrations of cycloheximide, cytosine arabinoside, tunicamycin, and 2-deoxy-D-glucose which effectively blocked cellular macromolecular synthesis did not significantly inhibit syncytium formation. These latter results suggested that de novo protein and DNA synthesis as well as protein glycosylation were not required for early syncytium formation. Thus, these experiments demonstrated that replication of BLV by the indicator cells was not essential for cell fusion.     

Kin17表达促进乳腺上皮细胞MCF-10A增殖

Kin17是一个与DNA复制、DNA修复有关的蛋白质,在人类的各种组织中表达均很低.乳腺上皮细胞生长增殖的分子机制尚未阐明.为了探讨Kin17与乳腺上皮细胞增殖的关系,检测了Kin17在不同增殖状况下的MCF-10A细胞中的表达情况,并把KIN17基因插入真核表达载体pCDNA3.1-(+)中,构建重组质粒pCDNA3.1-Kin17,通过转染MCF-10A细胞,检测Kin17的表达对MCF-10A细胞的增殖、DNA复制活性及信号分子表达的影响;同时在转染Kin17特异性小干扰RNA（siRNA_Kin17）后,分析MCF-10A细胞的Kin17表达及细胞生长状况.实验结果显示,经高浓度血清刺激后,细胞中Kin17表达升高,而且生长越快的细胞,Kin17表达越强;转染重组质粒pCDNA3.1-Kin17明显提高了MCF-10A细胞中Kin17的表达,同时Kin17的上调表达促进了细胞的增殖速度与DNA复制活性,增强了cyclin D1的表达水平.当转染siRNA_Kin17时使Kin17含量下调,MCF-10A细胞生长速度的抑制不显著.实验结果表明,Kin17与乳腺上皮细胞的DNA复制及生长增殖密切相关.对Kin17在乳腺上皮细胞增殖中的作用及分子调控机制的深入探讨,将有助于揭示乳腺癌细胞快速增殖的潜在机制.     

A human cytomegalovirus function inhibits replication of herpes simplex virus.

Human embryonic lung (HEL) cells infected with human cytomegalovirus (HCMV) restricted the replication of herpes simplex virus type 1 (HSV-1). A delay in HSV replication of 15 h as well as a consistent, almost 3 log inhibition of HSV replication in HCMV-infected cell cultures harvested 24 to 72 h after superinfection were observed compared with controls infected with HSV alone. Treatment of HCMV-infected HEL cells with cycloheximide (100 micrograms/ml) for 3 or 24 h, conditions known to result in accumulation of HCMV immediate-early and early mRNA, was demonstrated effective in blocking HCMV protein synthesis, as shown by immunoprecipitation with HCMV antibody-positive polyvalent serum. Cycloheximide treatment of HCMV-infected HEL cells and removal of the cycloheximide block before superinfection inhibited HSV-1 replication more efficiently than non-drug-treated superinfected controls. HCMV DNA-negative temperature-sensitive mutants restricted HSV as efficiently as wild-type HCMV suggesting that immediate-early and/or early events which occur before viral DNA synthesis are sufficient for inhibition of HSV. Inhibition of HSV-1 in HCMV-infected HEL cells was unaffected by elevated temperature (40.5 degrees C). However, prior UV irradiation of HCMV removed the block to HSV replication, demonstrating the requirement for an active HCMV genome. HSV-2 replication was similarly inhibited in HCMV-infected HEL cells. However, replication of adenovirus, another DNA virus, was not restricted in these cells under the same conditions. Superinfection of HCMV-infected HEL cells with HSV-1 labeled with [3H]thymidine provided evidence that the labeled virus could penetrate to the nucleus of cells after superinfection. Evidence for penetration of superinfecting HSV into HCMV-infected cells was also provided by blot hybridization of HSV DNA synthesized in cells infected with HSV alone versus superinfected cell cultures at 0 and 48 h after superinfection. In addition, superinfection with vesicular stomatitis virus ruled out a role for interferon in restriction of HSV replication in this system.     

Perturbations in simian virus 40 DNA synthesis by ultraviolet light.

Perturbations of Simian Virus 40 (SV40) DNA replication by ultraviolet (UV) light during the lytic cycle in permissive monkey CV-1 cells resemble those seen in host cell DNA replication. Formation of Form I DNA molecules (i.e. completion of SV40 DNA synthesis) was more sensitive to UV irradiation than synthesis of replicative intermediates or Form II molecules, consistent with inhibition of DNA chain elongation. The observed amounts of [3H]thymidine incorporated in UV-irradiated molecules could be predicted on the assumption that pyrimidine dimers are responsible for blocking nascent DNA strand growth. The relative proportion of labeled Form I molecules in UV-irradiated cultures rapidly increased to near-control values with incubation after 20 or 40 J/m2 of light (0.9--1.0 or 1.8--2.0 dimers per SV40 genome, respectively). This rapid increase and the failure of Form II molecules to accumulate suggest that SV40 growing forks can rapidly bypass many dimers. Form II molecules formed after UV irradiation were not converted to linear (Form III) molecules by the dimer-specific T4 endonuclease V, suggesting either that there are no gaps opposite dimers in these molecules or that T4 endonuclease V cannot use Form II molecules as substrates.